This application is a 371 of PCT/JP99/03691, filed on Jul. 8, 1999.
The present invention relates to LK6-A derivatives which have immmunosuppressive activity, cell growth inhibitory activity, anti-tumor activity, etc., and pharmaceutically acceptable salts thereof.
Cyclosporin A [Nature, Vol. 280, p. 148 (1978)], FK506 [Immunol. Today, Vol. 10, p. 6 (1989)], mizoribine [Transplantation Proceed., Vol. 11, p. 865, (1979)], azathioprine [New Eng. J. Med., Vol. 268, p. 1315 (1963)], 15-deoxyspergualin [Transplantation Proceed., Vol. 22, p. 1606 (1990)], etc., which are known as low-molecular immunosuppressive agents, are used as therapeutic agents for autoimmune diseases, allergic diseases, infections caused by organ transplantation, etc. or as rejection inhibitors in organ transplantation. However, they are not entirely satisfactory in respect of efficacy, side effect, etc.
Plakinidines [Tetrahedron Lett., Vol. 31, p. 3271 (1990)] are reported as compounds having the pyrrolo[4, 3, 2-de]quinoline skeleton, but their immunosuppressive activity has not been known. As the pyrrolo[4, 3, 2-de]quinoline compound having immunosuppressive activity, LK6-A represented by the following formula (Japanese Published Unexamined Patent Application No. 151185/97) has been reported. 
An object of the present invention is to provide novel LK6-A derivatives having excellent immunosuppressive activity, cell growth inhibitory activity, anti-tumor activity, etc. which are useful as therapeutic agents for autoimmune diseases, allergic diseases, and diseases caused by abnormal cell growth such as leukemia and cancers, or as rejection inhibitors in organ transplantation.
The present invention relates to LK6-A derivatives represented by general formula (I): 
[wherein R1 represents lower alkyl (the lower alkyl may be substituted by one to a substitutable number of, preferably 1-4 substituents which are the same or different and are selected from the group consisting of lower alkyl, hydroxy, lower alkoxy and halogen), lower alkanoyl (the lower alkyl moiety of the lower alkanoyl may be substituted by one to a substitutable number of, preferably 1-4 substituents which are the same or different and are selected from the group consisting of lower alkyl, hydroxy, lower alkoxy and halogen), carboxy, lower alkoxycarbonyl, 
(wherein n represents 1 or 2) or COCHxe2x95x90CHR9 {wherein R9 represents substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or NR10R11 (wherein R10 and R11, which may be the same or different, each represents hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl-substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl, or substituted or unsubstituted tetrahydropyranylmethyl, or R10 and R11 are combined together with the adjoining N to form a substituted or unsubstituted heterocyclic group)};
R2 represents hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy; halogen, SR12 (wherein R12 represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroaryl-substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl, or substituted or unsubstituted tetrahydropyranylmethyl), NR13R14 (wherein R13 and R14 have the same significances as the above R10 and R11, respectively) or azido;
R2xe2x80x2 represents hydrogen or is combined with R3 to represent a bond;
R3 represents substituted or unsubstituted lower alkanoyl or is combined with R2xe2x80x2 to represent a bond;
R4 and R5, which may be the same or different, each represents hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl, or substituted or unsubstituted heteroaryl-substituted lower alkoxycarbonyl;
R6 represents hydrogen or halogen; and
R7 and R8, which may be the same or different, each represents hydrogen, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower alkanoyl;
provided that a compound wherein R1 represents (E)-3-methoxyacryloyl, R2, R4, R5 and R6 represent hydrogen, R2xe2x80x2 and R3 are combined together to represent a bond, R7 represents hydrogen and R8 is acetyl is excluded], and pharmaceutically acceptable salts thereof.
Hereinafter, the compounds represented by general formula (I) are referred to as Compounds (I). The same shall apply to compounds of other formula numbers.
Preferred examples of the compounds of the present invention are shown in the following (a)-(h).
(a) Compound (I) in which R1 represents COCHxe2x95x90CHR9 (wherein R9 has the same significance as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R4, R5 and R6 represent hydrogen; and R7 and R8, which may be the same or different, each represents hydrogen or acetyl.
(b) Compound (I) in which R1 represents lower alkyl (the lower alkyl may be substituted by one to a substitutable number of, preferably 1-4 substituents which are the same or different and are selected from the group consisting of lower alkyl, hydroxy, lower alkoxy and halogen) or lower alkanoyl (the lower alkyl moiety of the lower alkanoyl may be substituted by one to a substitutable number of, preferably 1-4 substituents which are the same or different and are selected from the group consisting of lower alkyl, hydroxy, lower alkoxy and halogen); R2xe2x80x2 and R3 are combined together to represent a bond; R4, R5 and R6 represent hydrogen; and R7 and R8, which may be the same or different, each represents hydrogen or acetyl.
(c) Compound (I) in which R1 represents: 
(wherein n has the same significance as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R2, R4, R5 and R6 represent hydrogen; and R7 and R8, which may be the same or different, each represents hydrogen or acetyl.
(d) Compound (I) in which R1 represents (E)-3-methoxyacryloyl; R2xe2x80x2 and R3 are combined together to represent a bond; R4 represents hydrogen; and R5 represents substituted or unsubstituted lower alkoxycarbonyl or substituted or unsubstituted aralkyloxycarbonyl.
(e) Compound (I) in which R1 represents COCHR15CH(OCH3)2 (wherein R15 represents hydrogen or lower alkyl); R2xe2x80x2 and R3 are combined together to represent a bond; R4 and R5, which may be the same or different, each represents hydrogen or lower alkyl; and R7 and R8, which may be the same or different, each represents hydrogen, substituted or unsubstituted lower alkyl or acetyl.
(f) Compound (I) in which R1 represents COCHR15aCH(OCH3)2 (wherein R15a represents hydrogen or halogen); R2xe2x80x2 and R3 are combined together to represent a bond; R4 and R5 represent hydrogen; and R7, and R8, which may be the same or different, each represents hydrogen or acetyl.
(g) Compound (I) in which R1 represents 1-hydroxy-3-methoxypropyl; R2xe2x80x2 and R3 are combined together to represent a bond; R4 and R5 represent hydrogen; and R7 and R8, which may be the same or different, each represents hydrogen or acetyl.
(h) Compound (I) in which R2 represents hydrogen or substituted or unsubstituted lower alkanoyloxy; R2xe2x80x2 represents hydrogen; R3 represents substituted or unsubstituted lower alkanoyl; R4 represents hydrogen; R5 represents substituted or unsubstituted lower alkanoyl; R7 represents hydrogen; and R8 represents acetyl.
Pharmaceutically acceptable salts of Compounds (I) shown in the above (a)-(h) are also one of the preferred embodiments of the present invention.
In the definitions of the groups in Compounds (I), the halogen means a fluorine, chlorine, bromine or iodine atom.
The lower alkyl includes straight-chain or branched alkyl groups having 1-9 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl and nonyl.
The lower alkyl moiety of the lower alkanoyl, the lower alkoxy, the lower alkoxycarbonyl and the lower alkanoyloxy has the same significance as the above lower alkyl, and the lower alkyl moiety of the heteroaryl-substituted lower alkyl and the heteroaryl-substituted lower alkoxycarbonyl represents a group in which one hydrogen atom is removed from the above lower alkyl.
The lower alkenyl includes alkenyl groups having 2-6 carbon atoms, such as vinyl, 1-propenyl, butenyl, pentenyl and hexenyl, and the lower alkynyl includes alkynyl groups having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, pentynyl and hexynyl.
The aryl includes aryl groups having 6-14 carbon atoms, such as phenyl, naphthyl and anthryl, and the heteroaryl includes 5- or 6-membered heteroaryl groups, such as pyridyl, furyl, thienyl, pyrrolyl, imidazolyl, pyrimidinyl, oxazolyl, thiazolyl, bicyclic heteroaryl group such as indolyl, benzofuryl, benzothienyl, quinolyl, quinazolinyl and quinoxalinyl. The heteroaryl moiety of the heteroaryl-substituted lower alkyl and the heteroaryl-substituted lower alkoxycarbonyl has the same significance as the above heteroaryl. The aryl moiety of the aralkyl and the aralkyloxycarbonyl has the same significance as the above aryl. The alkylene moiety of the aralkyl and the aralkyloxycarbonyl represents a group in which one hydrogen atom is removed from the above lower alkyl.
The heterocyclic group formed with the adjoining N includes pyrrolidinyl, piperidino, piperazinyl, morpholino, thiomorpholino, pyrrolyl, imidazolyl and pyrazolyl.
The substituted lower alkyl, the substituted lower alkoxy, the substituted lower alkenyl, the substituted lower alkynyl, the substituted lower alkanoyl, the substituted lower alkanoyloxy, the substituted lower alkoxycarbonyl, the substituted aralkyloxycarbonyl, the substituted aralkyl, the substituted heteroaryl-substituted-lower alkyl and the substituted heteroaryl-substituted lower alkoxycarbonyl each has one to a substitutable number of, preferably 1-5 substituents which are the same or different. Examples of the substituents include NR16R17 (wherein R16 and R17, which may be the same or different, each represents hydrogen or lower alkyl, or R16 and R17 are combined together with the adjoining N to form a heterocyclic group), hydroxy, lower alkoxy and lower alkanoyloxy. The lower alkyl, the heterocyclic group formed with the adjoining N, the lower alkoxy and the lower alkanoyloxy have the same significances as defined above, respectively.
The substituted aryl and the substituted heteroaryl each has 1-3 substituents which are the same or different. Examples of the substituents include lower alkyl, NR16aR17a (wherein R16a and R17a have the same significances as the above R16 and R17, respectively), hydroxy, halogen, lower alkoxy, lower alkoxy-substituted lower alkoxy and lower alkanoyloxy. The lower alkyl, the lower alkoxy, the lower alkanoyloxy and the halogen have the same significances as defined above, respectively. The former lower alkoxy of the lower alkoxy-substituted lower alkoxy has the same significance as the above lower alkoxy, and the alkylene moiety of the latter lower alkoxy represents a group in which one hydrogen atom is removed from the above lower alkyl.
The substituted heterocyclic group formed with the adjoining N has 1-3 substituents which are the same or different. Examples of the substituents include hydroxy, lower alkyl, lower alkanoyl and arylcarbonyl. The lower alkyl and the lower alkanoyl have the same significances as defined above, respectively. The aryl moiety of the arylcarbonyl may be substituted by 1-3 functional groups arbitrarily selected from the group consisting of lower alkyl, lower alkanoyl, lower alkanoyloxy, hydroxy, lower alkoxy, amino, nitro, azido, carboxyl and lower alkoxycarbonyl. The alkyl moiety of the lower alkyl, lower alkanoyl, the lower alkanoyloxy, the lower alkoxy and the lower alkoxycarbonyl has the same significance as the above lower alkyl.
The substituted tetrahydropyranyl and the substituted tetrahydropyranylmethyl each has 1-4 substituents which are the same or different. Examples of the substituents include hydroxy, hydroxymethyl, lower alkoxy, lower alkoxymethyl, lower alkanoyloxy, lower alkanoyloxymethyl, benzyloxy, benzyloxymethyl and NR18R19 (wherein R18 and R19, which may be the same or different, each represents hydrogen, lower alkanoyl, lower alkoxycarbonyl, arylcarbonyl or aralkyloxycarbonyl). The lower alkyl moiety of the lower alkoxy, the lower alkoxymethyl, the lower alkanoyloxy, the lower alkanoyloxymethyl, the lower alkanoyl and the lower alkoxycarbonyl has the same significance as the above lower alkyl. The alkylene moiety of the aralkyloxycarbonyl has the same significance as the above alkylene moiety, and the aryl moiety of the arylcarbonyl and the aralkyloxycarbonyl has the same significance as the above aryl.
The pharmaceutically acceptable salts of Compounds (I) include acid addition salts, metal salts, ammonium salts, organic amine addition salts and amino acid addition salts. Examples of the acid addition salts are inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate and phosphate, and organic acid addition salts such as formate, acetate, oxalate, benzoate, methanesulfonate, p-toluenesulfonate, maleate, malonate, fumarate, tartrate, citrate, succinate and lactate. Examples of the metal salts are alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the ammonium salts are ammonium salt and tetramethylammonium salt. Examples of the organic amine addition salts are salts with morpholine and piperidine. Examples of the amino acid addition salts are salts with glycine, phenylalanine, aspartic acid, glutamic acid and lysine.
There may be various stereoisomers, regio isomers, geometrical isomers, tautomers, etc. for some of Compounds (I) of the present invention. The present invention encompasses all possible isomers and mixtures thereof in arbitrary mixture ratios.
The processes for preparing Compounds (I) are described below.
In the following processes, if the defined groups change under the conditions of the working method or are not appropriate for carrying out the method, the desired compounds can be obtained by using methods for introducing and eliminating protective groups which are conventionally used in synthetic organic chemistry [e.g., T. W. Greene, Protective Groups in Organic Synthesis, John Wiley and Sons Inc. (1981)]. If necessary, the order of the reaction steps such as introduction of a substituent may be changed.
Compound (Ia), i.e., Compound (I) wherein R1 represents acetyl; R2, R4, R5, R6, R7 and R8 represent hydrogen; and R2xe2x80x2 and R3 are combined together to represent a bond can be prepared according to the following reaction step. 
Step 1
Compound (Ia) can be obtained by treating LK6-A with an aqueous alkali solution in a solvent. Suitable solvents are water-miscible ones, for example, lower alcohols such as methanol and ethanol, tetrahydrofuran and dioxane, which may be used alone or as a mixture. As the aqueous alkali solution, 1-10 N aqueous solutions of alkalis such as sodium hydroxide and potassium hydroxide can be used. The reaction is carried out at a temperature between room temperature and the boiling point of the solvent used, preferably 50-100xc2x0 C. for 0.5-10 hours.
The processes for preparing Compound (II), i.e., Compound (I) wherein R1 represents COCHxe2x95x90CHR9 (wherein R9 has the same significance as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; and R4, R5 and R6 represent hydrogen are described in the following processes 2-7.
Compound (IIa), i.e., Compound (I) wherein R2, R4, R5 and R6 represent hydrogen; R2xe2x80x2 and R3 are combined together to represent a bond; R1 represents COCHxe2x95x90CHNR10R11 (wherein R10 and R11 have the same significances as defined above); R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reactions step. 
(In the formula, R10 and R11 have the same significances as defined above.)
Step 2
Compound (IIa) can be obtained by reaction of LK6-A with 1-20 equivalents of HNR10R11 (wherein R10 and R11 have the same significances as defined above) in an inert solvent. As the inert solvent, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, tetrahydrofuran, etc. may be used. The reaction is carried out at 0-100xc2x0 C., preferably 20-50xc2x0 C. for 0.5-12 hours.
Compound (IIb), i.e., Compound (I) wherein R1 represents COCHxe2x95x90CHNR10R11 (wherein R10 and R11 have the same significances as defined above), R2 represents NR13R14 (wherein R13 and R14 have the same significances as defined above, but NR13R14 here is the same as the above NR10R11); R4, R5 and R6 represent hydrogen; R2xe2x80x2 and R3 are combined together to represent a bond; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, R10, R11, R13 and R14 have the same significances as defined above, and NR13R14 is the same as NR10R11.)
Step 3
Compound (IIb) can be obtained by reaction of LK6-A with 2-100 equivalents of HNR10R11 (wherein R10 and R11 have the same significances as defined above) under the conditions similar to those in step 2.
Compound (IIc), i.e., Compound (I) wherein R1 represents (E)-3-methoxyacryloyl; R2 represents SR12 (wherein R12 has the same significance as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R4, R5 and R6 represent hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, R12 has the same significance as defined above.)
Step 4
Compound (IIc) can be obtained by reaction of LK6-A with 1-20 equivalents of HSR12 (wherein R12 has the same significance as defined above) in an inert solvent. The solvent, reaction temperature and reaction time are substantially the same as in the above step 2.
Compound (IId), i.e., Compound (I) wherein R1 represents (E)-3-methoxyacryloyl; R2 represents halogen; R2xe2x80x2 and R3 are combined together to represent a bond; R4, R5 and R6 represent hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, X1 represents halogen.)
The halogen represented by X1 has the same significance as the above halogen.
Step 5
Compound (IId) can be obtained by reaction of LK6-A with 1-20 equivalents of a halogenating reagent in an inert solvent.
As the inert solvent, halogen solvents such as dichloromethane, chloroform and carbon tetrachloride, ethers such as tetrahydrofuran and dioxane, lower alcohols such as methanol and ethanol, ethyl acetate, dimethylformamide, etc. may be used alone or as a mixture.
Examples of the halogenating reagent include bromine, chlorine, iodine, N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, tetrabutylammonium tribromide and pyrrolidone hydrotribromide. The reaction is carried out at a temperature between xe2x88x9220xc2x0 C. and the boiling point of the solvent used, preferably between 0xc2x0 C. and room temperature for 0.1-12 hours.
Compound (IIe), i.e., Compound (I) wherein R1 represents (E)-3-methoxyacryloyl; R2 represents hydrogen, halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R4, R5 and R6 represent hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
{In the formula, R2a represents hydrogen, halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above).}
The halogen represented by R2a has the same significance as the above halogen.
Step 6
Compound (IIe) can be obtained by heating Compound (IIIa) or (IIIb) obtained in the following process 8 or 9 in an inert solvent, if necessary, in the presence of molecular sieves. As the inert solvent, dimethyl sulfoxide, dimethylformamide, etc. may be used. The reaction is carried out at a temperature between 50xc2x0 C. and the boiling point of the solvent used, preferably 90-100xc2x0 C. for 1-120 hours.
Compound (IIf), i.e., Compound (I) wherein R1 represents (E)-COCHxe2x95x90CHAr (wherein Ar represents substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl which has the same significance as defined above); R2, R4, R5, R6, R7 and R8 represent hydrogen; and R2xe2x80x2 and R3 are combined together to represent a bond can be prepared according to the following reaction step. 
(In the formula, Ar represents substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl which has the same significance as defined above.)
Step 7
Compound (IIf) can be obtained by reaction of Compound (Ia) obtained in process 1 with 1-20 equivalents of an aldehyde represented by ArCHO (wherein Ar has the same significance as defined above) in an inert solvent in the presence of a base.
As the inert solvent, lower alcohols such as methanol and ethanol, ethers such as ether, tetrahydrofuran and dioxane, dimethylformamide, water, etc. may be used alone or as a mixture. As the base, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydride, potassium tert-butoxide, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc. may be used in an amount of 0.1-6 equivalents based on Compound (Ia). The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably between 0xc2x0 C. and room temperature for 1-240 hours.
The processes for preparing Compound (III), i.e., Compound (I) wherein R1 represents CR18R19CH2CH(OCH3)2 (wherein R18 represents hydrogen or is combined with R19 to represent xe2x95x90O, and R19 represents hydroxy or is combined with R18 to represent xe2x95x90O); R4, R5 and R6 represent hydrogen; and R2 and R3 are combined together to represent a bond are described in the following processes 8 and 9.
Compound (IIIa), i.e., Compound (III) wherein R18 and R19 are combined together to represent xe2x95x90O; R2 represents hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
Step 8
Compound (IIIa) can be obtained by reaction of LK6-A with 1-100 equivalents of methanol in an inert solvent, if necessary, in the presence of a base. As the inert solvent, halogen solvents such as dichloromethane and chloroform, ethers such as tetrahydrofuran and dioxane, dimethyl sulfoxide, dimethylformamide, etc. may be used. Methanol may be used also as the solvent. As the base, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, etc. may be used in an amount of 0.1-20 equivalents based on LK6-A. The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-60xc2x0 C. for 1-48 hours.
Compound (IIIb), i.e., Compound (III) wherein R18 and R19 are combined together to represent xe2x95x90O; R2 represents halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above); R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
{In the formula, R2b represents halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above).}
The halogen represented by R2b has the same significance as the above halogen.
Step 9
Compound (IIIb) can be obtained by subjecting Compound (IIIa) obtained in step 8 to the reaction similar to that in step 3, step 4 or step 5.
Compound (IIIa) and Compound (IIIb) obtained in step 8 and step 9 can be used as intermediates for further synthesizing novel derivatives. For example, Compound (IIIc), wherein R2b is converted into substituted or unsubstituted lower alkynyl (the lower alkynyl has the same significance as defined above), can be obtained by reaction of Compound (IIIba), i.e., the above Compound (IIIb) wherein R2b is bromine, with substituted or unsubstituted lower alkyne (the lower alkyne includes acetylene, propyne, butyne, pentyne and hexyne having 2-6 carbon atoms) in the presence of an appropriate palladium catalyst according to the method described in the literature [SYNTHESIS, p. 235 (1991)] or a similar method thereto. Further, Compound (IIId), wherein R2b is converted into substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, can also be obtained by reaction of Compound (IIIba) with various aromatic borate compounds or organic tin compounds instead of lower alkyne, which is known as the Suzuki reaction or the Stille reaction.
Compound (IIIe), wherein R2b is converted into substituted or unsubstituted lower alkyl, can be obtained by subjecting the above Compound (IIIc) to catalytic hydrogenation in an inert solvent in the presence of an appropriate catalyst. As the inert solvent, lower alcohols such as methanol and ethanol, ethyl acetate, dimethylformamide, etc. may be used alone or as a mixture. As the catalyst, any of the catalysts that are usually used in hydrogenation, for example, palladium/carbon and platinum oxide can be used. The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-30xc2x0 C. for 0.5-48 hours.
Compound (IIIf), wherein R2b is converted into substituted or unsubstituted lower alkenyl, can be obtained by using, as a catalyst, lead-treated palladium-calcium carbonate known as the Lindlar catalyst.
Any of these compounds wherein R1 represents COCH2CH(OCH3)2 can be converted into a compound wherein the carbonyl group in R1 is reduced, R18 represents hydrogen and R19 represents hydroxy by reducing the compound wherein R1 represents COCH2CH(OCH3)2 with 0.5-10 equivalents of sodium borohydride in an inert solvent. As the inert solvent, lower alcohols such as methanol and ethanol, dichloromethane, chloroform, dimethylformamide, etc. may be used alone or as a mixture. The reaction is carried out at a temperature between xe2x88x9220xc2x0 C. and the boiling point of the solvent used, preferably 0-30xc2x0 C. for 0.1-12 hours.
Compound (IV), i.e., Compound (I) wherein R1 represents: 
(wherein n has the same significance as defined above); R2, R4, R5 and R6 represent hydrogen; R2xe2x80x2 and R3 are combined together to represent a bond; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, n has the same significance as defined above.)
Step 10
Compound (IV) can be obtained by reaction of LK6-A with 1-100 equivalents of ethylene glycol or propylene glycol in an inert solvent, if necessary, in the presence of a base.
As the inert solvent, halogen solvents such as dichloromethane and chloroform, ethers such as tetrahydrofuran and dioxane, dimethyl sulfoxide, dimethylformamide, etc. may be used. Ethylene glycol or propylene glycol may be used also as the solvent.
As the base, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, etc. may be used in an amount of 0.1-20 equivalents based on LK6-A. The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-60xc2x0 C. for 1-96 hours.
Compound (V), i.e., Compound (I) wherein R1 represents (E)-3-methoxyacryloyl; R2 represents hydrogen; R2xe2x80x2 and R3 are combined together to represent a bond; R4 represents hydrogen; R5 represents substituted or unsubstituted lower alkoxycarbonyl or substituted or unsubstituted aralkyloxycarbonyl; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, R20 represents substituted or unsubstituted lower alkoxy or substituted or unsubstituted aralkyloxy.)
The substituted or unsubstituted lower alkoxy and substituted or unsubstituted aralkyloxy represented by R20 have the same significances as the above substituted or unsubstituted lower alkoxy and substituted or unsubstituted aralkyloxy, respectively.
Step 11
Compound (V) can be obtained by reaction of LK6-A with 1-5 equivalents of ClCOR20 (wherein R20 has the same significance as defined above) in an inert solvent in the presence of a base.
As the inert solvent, dichloromethane, chloroform, methanol, ethanol, dimethylformamide, etc. may be used alone or as a mixture.
As the base, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, etc. may be used in an amount of 1-5 equivalents based on LK6-A. The reaction is carried out at 0-50xc2x0 C. for 0.1-12 hours.
Compound (VI), i.e., Compound (I) wherein R1 represents COCHR15CH(OCH3)2 (wherein R15 represents lower alkyl); R2 represents hydrogen, halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R4 and R5, which may be the same or different, each represents hydrogen or lower alkyl; R7 represents hydrogen or lower alkyl; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, R4a and R5a, which may be the same or different, each represents hydrogen or lower alkyl; R7a represents hydrogen or lower alkyl; and R2a and R15 have the same significances as defined above.)
The lower alkyl represented by R4a, R5a and R7a has the same significance as the above lower alkyl.
Step 12
Compound (VI) can be obtained by reaction of Compound (IIIa) or (IIIb) with 1-10 equivalents of halogenated lower alkyl represented by R15bX2 (wherein R15b represents lower alkyl, and X2 represents halogen, and the lower alkyl represented by R15b and the halogen represented by X2 have the same significances as the above lower alkyl and halogen, respectively) in an inert solvent in the presence of 1-10 equivalents of a base.
Examples of the inert solvent include tetrahydrofuran, dioxane and dimethylformamide, and examples of the base include potassium carbonate, sodium hydride, potassium tert-butoxide and lithium diisopropylamide.
The reaction is carried out at a temperature between xe2x88x9278xc2x0 C. and the boiling point of the solvent used, preferably 0-30xc2x0 C. for 0.5-12 hours.
Compound (VII), i.e., Compound (I) wherein R1 represents COCX3HCH(OCH3)2 (wherein X3 represents halogen, and the halogen represented by X3 has the same significance as the above halogen); R2 represents hydrogen, halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above); R2xe2x80x2, and R3 are combined together to represent a bond; R4 and R5 represent hydrogen; R6 represents hydrogen or halogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, X3, R6 and R2a have the same significances as defined above.)
Step 13
Compound (VII) can be obtained by reaction of Compound (IIIa) or (IIIb) with 1-10 equivalents of a halogenating reagent in an inert solvent, if necessary, in the presence of 1-10 equivalents of a base.
Examples of the base include triethylamine, diisopropylethylamine, potassium carbonate and sodium carbonate. Examples of the halogenating reagent include bromine, chlorine, iodine, N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, tetrabutylammonium tribromide and pyrrolidone hydrotribromide.
As the inert solvent, dichloromethane, chloroform, carbon tetrachloride, methanol, ethanol, tetrahydrofuran, dioxane, dimethylformamide, etc. may be used alone or as a mixture. The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-30xc2x0 C. for 0.5-24 hours.
Compound (VIII), i.e., Compound (I) wherein R1 represents 1-hydroxy-3-methoxypropyl; R2 represents hydrogen, halogen, SR12 (wherein R12 has the same significance as defined above) or NR13R14 (wherein R13 and R14 have the same significances as defined above); R2xe2x80x2 and R3 are combined together to represent a bond; R4 and R5 represent hydrogen; R6 represents hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
(In the formula, R2a has the same significance as defined above.)
Step 14
Compound (VIII) can be obtained by reducing LK6-A or Compound (IIe) with 1-10 equivalents of sodium borohydride in an inert solvent.
As the inert solvent, lower alcohols such as methanol and ethanol, dichloromethane, chloroform, dimethylformamide, etc. may be used alone or as a mixture.
The reaction is carried out at a temperature between xe2x88x9220xc2x0 C. and the boiling point of the solvent used, preferably 0-30xc2x0 C. for 0.1-12 hours.
Compound (IX), i.e., Compound (I) wherein R1 represents (E)-3-methoxyacryloyl or COCH2CH(OCH3)2; R2 represents hydrogen or substituted or unsubstituted lower alkanoyloxy; R2xe2x80x2 represents hydrogen; R3 represents substituted or unsubstituted lower alkanoyl; R4 represents hydrogen; R5 represents substituted or unsubstituted lower alkanoyl; R6 represents hydrogen; R7 represents hydrogen; and R8 represents acetyl can be prepared according to the following reaction step. 
[In the formula, R1a represents (E)-3-methoxyacryloyl or COCH2CH(OCH3)2; R2c represents hydrogen or substituted or unsubstituted lower alkanoyloxy; and R21 represents substituted or unsubstituted lower alkyl.]
The substituted or unsubstituted lower alkanoyloxy represented by R2c has the same significance as the above substituted or unsubstituted lower alkanoyloxy, and the substituted or unsubstituted lower alkyl represented by R21 has the same significance as the above substituted or unsubstituted lower alkyl.
Step 15
Compound (IX) can be obtained by reaction of LK6-A or Compound (IIIa) with 2-100 equivalents of an acid anhydride, if necessary, in an inert solvent.
Examples of the inert solvent include dichloromethane, chloroform and dimethylformaimde, and the acid anhydride may be used also as the solvent. The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-30xc2x0 C. for 1-72 hours.
Further conversion of R2c is possible using Compound (IX) obtained in step 15 as a synthetic intermediate. For example, Compound (IXa), i.e., Compound (IX) wherein R2c is hydrogen can be obtained by hydrogenating the compound wherein R2c is lower alkanoyloxy in an inert solvent in the presence of an appropriate catalyst. As the inert solvent, lower alcohols such as methanol and ethanol, ethyl acetate, dimethylformamide, etc. may be used alone or as a mixture. Appropriate catalysts include those conventionally used in hydrogenation, for example, palladium/carbon and platinum oxide.
The reaction is carried out at a temperature between 0xc2x0 C. and the boiling point of the solvent used, preferably 20-30xc2x0 C. for 0.5-48 hours.
The above Compounds (I)-(IX) can be obtained by appropriately combining the above-described methods. Further, Compounds (I) described in the present invention can be obtained by combining methods conventionally used in synthetic organic chemistry.
The desired compounds in the processes described above can be purified by appropriate combinations of purification methods conventionally used in synthetic organic chemistry, for example, filtration, extraction, washing, drying, concentration, crystallization and various kinds of chromatography. The intermediates may be subjected to the subsequent reaction without purification.
In the case where a salt of Compound (I) is desired and it is produced in the form of the desired salt, it can be subjected to purification as such. In the case where Compound (I) is produced in the free state and its salt is desired, the salt can be formed according to a conventional method, that is, by dissolving or suspending Compound (I) in a suitable solvent and adding a desired acid or base thereto.
Compounds (I) and pharmaceutically acceptable salts thereof may exist in the form of adducts with water or various solvents, which are also within the scope of the present invention.
Compounds (I) and pharmaceutically acceptable salts thereof can be used as such or in various pharmaceutical forms according to the pharmacological activity and the purpose of administration. Pharmaceutical compositions of the present invention can be prepared by uniformly mixing an effective amount of Compound (I) or a pharmaceutically acceptable salt thereof, as an active ingredient, with a pharmaceutically acceptable carrier. The carrier can take a wide variety of forms according to the pharmaceutical form desirable for administration. These pharmaceutical compositions are preferably in a unit dose form suitable for oral administration or parenteral administration in the form of ointment, injection, or the like.
Tablets can be prepared using excipients such as lactose, glucose, sucrose, mannitol and methyl cellulose, disintegrating agents such as starch, sodium alginate, calcium carboxymethyl cellulose and crystalline cellulose, lubricants such as magnesium stearate and talc, binders such as gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose and methyl cellulose, surfactants such as sucrose fatty acid ester and sorbitol fatty acid ester, and the like in a conventional manner. It is preferred that each tablet contains 1-300 mg of the active ingredient.
Granules can be prepared using excipients such as lactose and sucrose, disintegrating agents such as starch, binders such as gelatin, and the like in a conventional manner. Powders can be prepared using excipients such as lactose and mannitol, and the like in a conventional manner. Capsules can be prepared using gelatin, water, sucrose, gum arabic, sorbitol, glycerin, crystalline cellulose, magnesium stearate, talc, and the like in a conventional manner. It is preferred that each capsule contains 1-300 mg of the active ingredient.
Syrup can be prepared using sugars such as sucrose, water, ethanol, and the like in a conventional manner.
Ointment-can be prepared using ointment bases such as vaseline, liquid paraffin, lanolin and macrogol, emulsifiers such as sodium lauryl lactate, benzalkonium chloride, sorbitan mono-fatty acid ester, sodium carboxymethyl cellulose and gum arabic, and the like in a conventional manner.
Injections can be prepared using solvents such as water, physiological saline, vegetable oils (e.g., olive oil and peanut oil), ethyl oleate and propylene glycol, solubilizing agents such as sodium benzoate, sodium salicylate and urethane, isotonicity agents such as sodium chloride and glucose, preservatives such as phenol, cresol, p-hydroxybenzoic acid ester and chlorobutanol, antioxidants such as ascorbic acid and sodium pyrosulfite, and the like in a conventional manner.
Compounds (I) and pharmaceutically acceptable salts thereof can be administered orally or parenterally as an ointment, injection, or the like. The effective dose and the administration schedule of Compound (I) or a pharmaceutically acceptable salt thereof will vary depending on the mode of administration, the patient""s age, body weight and condition, etc. However, it is generally preferred to administer Compound (I) or a pharmaceutically acceptable salt thereof in a dose of 0.01-20 mg/kg 1-4 times a day.
Examples of Compounds (I) obtained by the present invention are shown in Tables 1 and 2.
The immunosuppressive activity of typical Compounds (I) is described below.
Lymph node was aseptically excised from a B10.BR mouse (Japan SLC Inc.) and washed with a solution comprising Hanks"" balanced salt solution (HBSS, Gibco) and 2.5% fetal calf serum (FCS, Gibco) (HBSS-FCS). To the washed lymph node was added RPMI1640 medium comprising 10% FCS, 1% 200 mM L-glutamine, a 1% penicillin-streptomycin solution, 5% NCTC-109, 1% 1 M HEPES (all produced by Gibco), 7.5% sodium hydrogen carbonate and 0.1% 50 mM 2-mercaptoethanol (hereinafter referred to as RPMI1640-FCS) to prepare a single cell suspension having a density of 3xc3x97106 cells/ml.
Separately, spleen was aseptically excised from an AKR mouse (Japan SLC Inc.) to prepare a single cell suspension with HBSS-FCS. To the obtained cell suspension was added mitomycin C (MMC) (Kyowa Hakko Kogyo Co., Ltd.) to a final concentration of 0.05 mg/ml, followed by incubation at 37xc2x0 C. for 30 minutes. Then, the suspension was washed three times with HBSS-FCS, and a single cell suspension having a density of 1xc3x97107 cells/ml was prepared using RP1640-FCS.
Into each well of a 96-well microtiter plate were put 0.05 ml of the B10. BR mouse lymph node cell suspension (containing 1.5xc3x97105 cells), 0.05 ml of the AKR mouse spleen cell suspension (containing 5xc3x97105 cells) and 0.1 ml of a solution of Compound (I) in RPMI1640-FCS at each test concentration, followed by incubation in a CO2 incubator at 37xc2x0 C. for 72 hours. The solutions of the test compound were prepared to give final concentrations of 7xc3x9710xe2x88x9210-7xc3x9710xe2x88x926 M.
[3H]-Thymidine was added to the wells in an amount of 1xc3x9710xe2x88x926 Ci, 18 hours before the end of incubation. After the incubation, the cells were collected on filter paper with a cell harvester, followed by drying. A toluene scintillator was added to the cells, and the radioactivity of [3H]-thymidine incorporated into the cells was determined using a liquid scintillation counter (test group).
As a control group, 0.1 ml of RPMI1640-FCS containing no test compound was added, followed by incubation in the same manner as above, and the radioactivity of [3H]-thymidine incorporated into the cells was determined. To 0.05 ml of the B10.BR mouse lymph node cell suspension (containing 1.5xc3x97105 cells) or 0.05 ml of the AKR mouse spleen cell suspension (containing 5xc3x97105 cells) was added 0.15 ml of RPMI1640-FCS, followed by incubation in the same manner as above, and the radioactivity of [3H]-thymidine incorporated into the cells was determined.
The T cell growth inhibition rate was calculated according to the following equation.
T cell growth inhibition rate (%)=(Cxe2x88x92T)/{Cxe2x88x92(A+B)}xc3x97100
C: Radioactivity of the control group
T: Radioactivity of the test group
A: Radioactivity of the MMC-treated AKR mouse
B: Radioactivity of the B10.BR mouse
(In the equation, the radioactivity of the MMC-treated AKR mouse refers to the radioactivity of [3H]-thymidine incorporated, into the MMC-treated AKR mouse spleen cells, and the radioactivity of the B10.BR mouse refers to the radioactivity of [3H]-thymidine incorporated into the B10.BR mouse lymph node cells.)
The 50% inhibitory concentration of each compound against the growth of T cells in mixed mouse lymphocyte reaction was calculated from the above equation. The results are shown in Table 3.
Balb/c strain male mice (8-weeks-old, Charles River) were immunized by subcutaneous administration of 0.1 ml of 2,4,6-trinitrobenzene sulfonic acid (TNBS) (adjusted to 10 mM with a phosphate buffer) into the right side. The test was carried out using groups of mice, each group consisting of 5 animals, which are a control group treated with 0.3% methyl cellulose containing 3% DMSO, a group treated with a fixed concentration of a test compound suspended in 0.3% methyl cellulose containing 3% DMSO, and a group treated with cyclosporin A (Sandoz Pharmaceuticals, Ltd.).
The 0.3% methyl cellulose containing 3% DMSO or the test compound was intraperitoneally administered to the mice 30 minutes before the immunization treatment and thereafter every 24 hours, totally 5 times. Cyclosporin A was orally administered one hour before the immunization treatment and thereafter every 24 hours, totally 5 times. On the fifth day when sensitization was established, 0.05 ml of the above 10 mM TNBS as a causative antigen was subcutaneously injected into the right hind sole. Eighteen hours after the injection, the thickness of both feet of each mouse of the groups treated with respective amounts of test compound was measured with Dial Thickness Gauge. The value (T) was obtained by subtracting the thickness of the left foot from that of the right foot. Separately the thickness of both feet of each mouse of the group treated with no test compound was measured and the value (C) was obtained by subtracting the thickness of the left foot from that of the right foot. The suppression rate (%) was determined according to the equation [(Cxe2x88x92T)/C]xc3x97100 (%). The results are shown in Table 4.
As can be seen from Tables 3 and 4, Compounds (I) have an excellent immunosuppressive activity and are useful as therapeutic agents for autoimmune diseases, allergic diseases, infections caused by organ transplantation, etc. Compounds (I) are also useful as therapeutic agents for diseases caused by abnormal cell growth such as leukemia and cancers.